Methylphenobarbital

In the method described here, an aliquot of a urine sample is analyzed to detect barbiturates through dilution and ultra-high-performance chromatography-tandem mass spectrometry (UPLC-MS/MS) using deuterated internal standards. This assay detects the presence of nine barbiturate drugs-amobarbital, barbital, butalbital, butabarbital, mephobarbital, secobarbital, pentobarbital, phenobarbital, and thiopental. This protocol describes two LC separation methods-first LC method (2.2 min/sample) is intended to be used as a first step of the analysis that does not separate amobarbital and pentobarbital, and a second, longer (2.7 min/sample) LC method is intended to be used only for samples which have a peak in the amobarbital/pentobarbital retention time on the shorter LC method. Since the frequency at which amobarbital and pentobarbital are observed in clinical populations is low, the shorter LC method helps gain efficiency in a high-volume laboratory environment. Additional features of this protocol that help in efficiency gain are automated extraction using Hamilton™ liquid handling system and algorithmic data review using Ascent™ software.

Barbiturates which are old pharmaceutical drugs are still widely used in medical treatment of epilepsy and for general anesthesia. To date, more than 2500 different barbituric acid analogs have been synthesized, and 50 of them were introduced into medical use over the last century. Due to their highly addictive properties, pharmaceuticals containing barbiturates are under strict control in many countries. However, by considering the worldwide problem with new psychoactive substances (NPS) the introduction of new designer barbiturate analogs into the dark market might serve a serious public health problem in the near future. For this reason there is an increasing need for application methods for barbiturates monitoring in biological samples. The UHPLC-QqQ-MS/MS method for determination of 15 barbiturates, phenytoin, methyprylon and glutethimide was developed and fully validated. The biological sample volume was reduced to only 50 µL. A simple LLE (pH 3 with ethyl acetate) was successfully applied. The lower LOQ was 10 ng/mL. The method enables differentiation of structural isomers: hexobarbital and cyclobarbital; as well as amobarbital and pentobarbital. Chromatographic separation was achieved with the use of the alkaline mobile phase (pH 9) and Acquity UPLC BEH C18 column. Furthermore, the novel fragmentation mechanism of barbiturates was proposed, which may have a great impact in identification of novel barbiturates analogs introduced to illegal marketplaces. The presented technique has a great potential to be applied in forensic, clinical and veterinary toxicological laboratories, as was evidenced by the positive results of international proficiency tests.